Most children diagnosed with neuroblastoma after infancy have a poor prognosis, and few will survive despite intensive therapy. New treatments are urgently needed. Recently, drugs which block blood vessel growth (angiogenesis) into tumors have been clinically validated in adults, and may offer promise for these patients. One validated anti-angiogenic target is vascular endothelial growth factor (VEGF), which is expressed in essentially all human cancers, including neuroblastoma. VEGF can bind to both VEGF-receptor-1 (VEGFR1) and VEGFR2, but much prior work indicates that VEGF stimulates angiogenesis primarily via VEGFR2. Therefore, antagonism of VEGF/VEGFR2 signaling has been the principal approach tested against experimental and human cancers. We have previously demonstrated that drugs which block VEGF and VEGFR2 lessen angiogenesis and growth of experimental early-stage neuroblastoma tumors, suggesting that this therapy may be therapeutically useful in children with this cancer. However, our experiments also demonstrate that these tumors ultimately become resistant to VEGF/VEGFR2 blockade. These results suggest that an alternative mechanism may support angiogenesis in neuroblastoma when VEGF/ VEGFR2 signaling is blocked. In these studies, we will determine if activation of VEGFR1 and Notch pathways in neuroblastoma tumor vessels constitutes one such alternative. Both VEGFR1 and Notch proteins are known to contribute to angiogenesis in disease states. In addition, activation of each of these two pathways may stimulate the other. Our initial experiments support this concept, and demonstrate VEGFR1 and Notch activation in the vessels of neuroblastoma tumors that are resistant to anti-VEGF treatment. These observations lead us to propose that one mechanism by which neuroblastomas compensate for anti-VEGF treatment is to preserve tumor blood supply by activating these alternative pathways in vasculature. Utilizing our well-characterized xenograft model, in which NB cell lines are implanted in the kidney of athymic mice, and the TH-MYCN genetic model of NB, we will, in Aim 1, determine whether VEGFR1 signaling can rescue tumor vasculature from VEGFR2 blockade; and in Aim 2, we will determine whether Notch activation in vessels promotes tumor resistance to VEGFR2 blockade. Our overall goal in these studies is to develop effective new treatments children with neuroblastoma by identifying and then overcoming the mechanisms by which these tumors evade inhibition of VEGF. PUBLIC HEALTH RELEVANCE: Children with metastatic neuroblastoma have a poor prognosis with only 30-35% surviving despite intensive therapy. Drugs which block blood vessel growth (angiogenesis) into tumors have been clinically validated in adults, and may offer promise for these patients. Our overall goal in these studies is to develop an effective anti-angiogenic therapy for children with neuroblastoma by identifying and then overcoming the mechanisms by which these tumors evade VEGF-blocking therapies.